Lactic acid is widely used in the food, pharmaceutical, plastics and textile industries. It is also used as a source of lactic acid polymers which find utility as biodegradable plastics and of which the physical properties can be controlled by adjusting the proportions of the L(+)- and D(−)-lactides.
Lactic acid can be produced by fermentation but the economics of such production is strongly dependent upon the cost of the raw materials. It is, for instance, very expensive when refined sugars and starch are used as the fermentation feedstock. Lignocellulosic biomass, which has no competing food value, is a lower-cost, alternative feedstock having wide availability and the potential to be sourced sustainably. However, there is still a need in the art to improve the commercial scale fermentation of lignocellulosic biomass for lactic acid production.
The present invention is concerned in part with methods of treatment of lignocellulosic materials which render the materials more amenable to biologically mediated transformations. More particularly, it is concerned with methods of treatment which render the lignocellulosic materials more amenable to at least one of: enzymatic hydrolysis of carbohydrate components to sugars by saccharolytic enzymes present in the pretreated biomass; microbial hydrolysis by microorganisms capable of the fermentation of hexose sugars such as glucose, mannose, and galactose; and, microbial hydrolysis by microorganisms capable of the fermentation of pentose sugars such as xylose and arabinose.
Approximately 90% of the dry weight of most plant materials is stored in the form of cellulose, hemicellulose, lignin and pectin, with the remainder being constituted by proteins, ash and extractives such as non-structural sugars, nitrogenous materials, chlorophyll and waxes.
Cellulose is the main structural constituent in plant cell walls. It exists mainly in a crystalline form and is typically found in an organized fibrous structure: the linear cellulose polymer consists of D-glucose subunits linked to each other by β-(1,4)-glycosidic bonds; cellobiose is the repeat unit established through this linkage, and it constitutes cellulose chains; in turn, the long-chain cellulose polymers are linked together by hydrogen and van der Waals bonds, which cause the cellulose to be packed into microfibrils; and, hemicelluloses and lignin then cover said microfibrils. Fermentable D-glucose can be produced through the action of either acid or enzymes breaking the β-(1,4)-glycosidic linkages and the amorphous form of cellulose is more susceptible to such enzymatic degradation. However, high cellulose crystallinity, low accessible surface area, protection by lignin, and sheathing by hemicellulose all contribute to the resistance of cellulose in lignocellulosic biomass to hydrolysis.
Hemicellulose is mainly differentiated from cellulose in that hemicellulose has branches with short lateral chains consisting of different sugars. These monosaccharides include pentoses (xylose, rhamnose, and arabinose), hexoses (glucose, mannose, and galactose), and uronic acids (e.g., 4-o-methylglucuronic, D-glucuronic, and D-galactouronic acids). The backbone of hemicellulose is either a homopolymer or a heteropolymer with short branches linked by β-(1,4)-glycosidic bonds and occasionally β-(1,3)-glycosidic bonds. Also, hemicelluloses can have some degree of acetylation.
Lignin is a complex, three-dimensional polymer constituted by phenylpropanoid subunits linked together by a variety of ether and carbon-carbon bonds. Lignin is intimately interlaced with hemicelluloses in the plant cell wall forming a matrix to cover the crystalline cellulose microfibrils. Whilst it imparts structural support and impermeability to the cell wall, its presence concomitantly provides a protective barrier that prevents plant cell destruction by fungi and those bacteria necessary for the conversion of biomass to organic acids. Lignin's aromatic nature and complex structure make lignin degradation very difficult. Both lignin and lignin-derived compounds have a detrimental effect on the enzymatic hydrolysis of biomass because they physically hinder the accessibility of cellulases; they also bind cellulases and lead to their inactivation.
Pre-treatment methods to break down lignin are thus essential for the effective enzymatic and microbial hydrolysis of lignocellulose and thus for the conversion of lignocellulose into organic acids such as lactic acid, succinic acid and acetic acid. Known pre-treatment methods can be roughly divided into different categories: physical (milling and grinding), physicochemical (steam pre-treatment/auto-hydrolysis, hydro-thermolysis, and wet oxidation), chemical (alkali, dilute acid, oxidizing agents, and organic solvents), biological, electrical, or a combination of these. The present invention is concerned with a chemical pre-treatment process utilizing an alkaline agent.
Compared with acid pre-treatment processes, alkaline processes are considered to cause less sugar degradation, and many of the caustic salts can be recovered and/or regenerated. Kong et al. Effects of cell-wall acetate, xylan backbone, and lignin on enzymatic hydrolysis of aspen wood, Appl. Biochem. Biotechnol. 1992, 34/35, 23-35 reported that alkalis remove acetyl groups from hemicellulose (mainly xylan), thereby reducing the steric hindrance of hydrolytic enzymes and greatly enhancing carbohydrate digestibility.
Historically, sodium, potassium, calcium, and ammonium hydroxides have been preferred as alkaline pre-treatment agents and, of these, sodium hydroxide has been the most studied, as documented in, for instance: Fox, D. J et al., Comparison of alkali and steam (acid) pretreatments of lignocellulosic materials to increase enzymic susceptibility: Evaluation under optimized pretreatment conditions J. Chem. Tech. Biotech. 1989, 44, 135-146; and, MacDonald, D. G. et al. Alkali treatment of corn stover to improve sugar production by enzymatic hydrolysis Biotechnol. Bioeng. 1983, 25, 2067-2076.
Calcium hydroxide (slake lime) has also found utility as a pre-treatment agent, mainly on account of the facts that it is relatively inexpensive (per kilogram) and that it is possible to recover calcium from an aqueous reaction system as insoluble calcium carbonate by neutralizing it with inexpensive carbon dioxide; the calcium hydroxide can subsequently be regenerated using established lime kiln technology. Lime pre-treatment does however tend to increase the crystallinity index of the pre-treated lignocellulosic biomass. Whilst this may not have an effect on ultimate sugar yields from enzymatic hydrolysis, the crystallinity significantly affects initial hydrolysis rates as reported in Chang et al. Fundamental factors affecting biomass enzymatic reactivity, Appl. Biochem. Biotechnol. 2000, 84-86, 5-37.
Further as reported by Kim et al. Effect of structural features on enzyme digestibility of corn stover, Bioresour. Technol. 2006, 97, 583-591, the delignification of a given lignocellulosic material with calcium hydroxide can vary significantly with oxidative conditions and temperature. This brings into question the efficacy of calcium hydroxide in industrial processes where oxidative conditions cannot easily be moderated and where the lignocellulosic feedstock may be derived from more than one plant material or source, noting that the composition of lignin, hemicellulose and cellulose can vary from one plant species to another and, for a single plant type may vary with age and stage of growth.
WO2013/062407 (Wageningen University et al.) describes a process for the conversion of lignocellulosic material into an organic acid comprising an alkaline pre-treatment step and a fermentation step. Whilst the document purports magnesium oxide or magnesium hydroxide could be used in the alkaline pre-treatment step, this is not exemplified. Rather this document only demonstrates the use of calcium oxide or calcium hydroxide in a pre-treatment step which occurs at a temperature of from 20° to 115° C. For the purposes of achieving water balance in the process, the liquid phase obtained in the fermentation step must be recycled to the alkaline pre-treatment and/or the fermentation step.